PURIFICATION AND PARTIAL CHARACTERIZATION OF ...

Received: April 3, 2003 J. Venom. Anim. Toxins incl. Trop. Dis. Accepted: October 10, 2003 V.10, n.3, p.242-259, 2004. Published online: September 10, 2004 Original paper - ISSN 1678-9199.

PURIFICATION AND PARTIAL CHARACTERIZATION OF PHOSPHOLIPASES

A2 FROM Bothrops asper (BARBA AMARILLA) SNAKE VENOM FROM

CHIRIGUANÁ (CESAR, COLOMBIA)

RAMÍREZ-AVILA J.1, QUEVEDO B. E.2, LÓPEZ E.3, RENJIFO J. M.1

1 Serum Group, National Institute of Health (INS), Bogotá, Colombia, South America; 2 Faculty of

Engineering, National University of Colombia, Bogotá, Colombia, South America; 3 Department of

Chemistry, Faculty of Sciences, National University of Colombia, Bogotá, Colombia, South America.

ABSTRACT. Components with phospholipase A2 activity were isolated by gel filtration and

cationic exchange chromatography from the venom of Bothrops asper snakes from

Chiriguaná, Colombia (9°22´N; 73°37´W). Five fractions were obtained by the gel filtration,

and PLA2 activity was found in fraction 3 (F3). In the cationic exchange chromatography, F3

showed eight components with PLA2 activity. Six of these components appeared as one band

in polyacrylamide gel electrophoresis (SDS-PAGE). Fractions II and VII exhibited an optimal

activity at pH 9 and 52ºC. The optimum calcium concentration for fraction II was 48 mM and

for fraction VII, 384 mM. Both fractions showed thermal stability. Fraction II was stable at

pH values between 2.5 and 9, and fraction VII, between 2.5 and 8. The Michaelis Menten

constant (KM) was 3.5x10-3 M for fraction II and 1.6x10-3 M for fraction VII. The molecular

weight was 16,000 Dalton for fraction II and 17,000 Dalton for fraction VII. Both isoenzymes

did not show any toxic activity (DL50) at 5.3 and 4 µg/g. The two fractions showed different

kinetic constant (KM), calcium requirement, and substrate specificity for haemolytic activity.

KEY WORDS: phospholipase, isoenzymes, snake, venom, Bothrops asper, Colombia

CORRESPONDENCE TO: J. Ramírez-Avila, Avenida calle 26, N° 51-60, Bogotá, D. C.,

Colombia, South America. Phone: (571) 220-77-00, Ext. 485. Fax: (571) 222-34-68.

Email: [email protected]

mailto:[email protected]

J. Ramírez-Avila et al. PURIFICATION AND PARTIAL CHARACTERIZATION OF PHOSPHOLIPASES A2 FROM Bothrops asper (BARBA AMARILLA) SNAKE VENOM FROM CHIRIGUANÁ (CESAR, COLOMBIA). J. Venom. Anim. Toxins incl. Trop. Dis., 2004, 10, 3, p.243.

INTRODUCTION

Phospholipases A2 (PLA2s) (EC.3.1.1.4) catalyzes the hydrolysis of the 2-acyl ester linkage of

1,2-diacyl-3-sn-phosphoglycerides, with Ca2+ requirement (20,25). These enzymes play an

important role in the lipid metabolism, and have been widely used to study the structure of

lipoproteins (1) among other applications.

Snake venoms are particularly rich in PLA2s (5,12,15,22) and some of them contain more

than one isoform of this enzyme. Many of these isoenzymes have similar molecular weights

but can be differentiated in a small number of amino acids (9,20) by ion-exchange

chromatography (14,15,26) or isoelectric focusing (23). Snake venom PLA2s are able to

induce several biological effects, such as pre-synaptic or post-synaptic neurotoxicity,

cardiotoxicity, myotoxicity, platelet aggregation, oedema formation, haemolysis,

anticoagulation, convulsion, and hypotension (26). This diversity of physiological functions

of PLA2s isoenzymes from snake venoms is very important for the production of antivenoms

and to understand the accelerated evolution of this enzyme in the Viperidae venom (19,20).

Therefore, it is also very important to identify the diversity of isoforms present in the same

species and in different populations.

In Colombia, snakes of the Bothrops genus have medical importance since they are

responsible for more than 90% of the total snakebites (unpublished information from INS).

Bothrops asper (Family Viperidae, Subfamily Crotalinae) is the only species of that genus

found in northern Colombia. It is poorly differentiated from Bothrops atrox, and there is a

great controversy upon the taxonomic status of these two species (2). Snakes of the Bothrops

genus from northern Colombia are classified as B. asper, based on the data published by

Campbell and Lamar (2).

In the present study, we report the isolation and partial characterization of snake venom

fractions with phospholipases A2 activity from B. asper snakes from Chiriguaná (Cesar),

northern Colombia.


MATERIALS AND METHODS

Venom

B. asper venom was obtained from several specimens collected in Chiriguaná (Cesar,

Colombia). Venom was filtered, lyophilized and stored at -20°C in the venom bank of the

Instituto Nacional de Salud, Bogotá, D. C., Colombia.

Isolation of phospholipase A2

Crude venom (100 mg) was dissolved in 1 ml of 5% acetic acid, cleared by centrifugation for

30 minutes at 1,800 g, submitted to gel filtration on Sephadex G-75 (Pharmacia LKB,

Sweden) column (2.6 x 71.5 cm), and then, eluted with the same solvent. Fractions of 10 ml

were collected at a flow rate of 60 ml/h using a Frac-200 fraction collector (Pharmacia LKB).

F3 with PLA2 activity was taken up from the gel filtration and applied to a Sephadex G-25

(Pharmacia LKB) column (1.6 x 14.5 cm) previously equilibrated, and then, eluted with 10

mM of acetic acid, pH 5.

F3 in 10 mM of acetic acid, pH 5 was applied to ion-exchange chromatography on a MONO

S (HR 5/5) column (Pharmacia LKB), which had been previously equilibrated with a similar

solvent. The elution used a constant concentration gradient, from 0 to 1 M of NaCl with the

same solvent. Fractions of 1 ml were collected at 60 ml/h. The separation was done on FPLC

(Pharmacia LKB); the absorbances at 280 nm, conductivities and collected fractions were

monitored. The resulting eight peaks were collected, pooled, and dialyzed using membranes

(Spectra/Por 1) with a molecular weight of 6,000-8,000 Da, and later lyophilized. An aliquot

of dialyzed fractions was used for the determination of protein concentration by the Lowry

method (13).

Polyacrylamide gel electrophoresis (SDS-PAGE)

Gel for SDS-PAGE was prepared and run using a Hoeffer minigel electrophoresis system.

Electrophoresis was performed in presence of sodium dodecyl sulfate (SDS) in 0.75 mm thick

slab gels, according to the Laemmli method (10). The gels were silver stained (17). The

mobility of standard proteins (14.4-94 KDa) and molecular weights of the samples were

analysed using the Kodak Digital Science 1D Image Analysis Software (4).


Lethality assays

Lethality assays were performed with ICR male mice (16-18 g) by intraperitoneal (i.p).

injection with 0.2 ml of the tested solution. The LD50 was evaluated 48 hours after the

injection (21).

Phospholipase A2 activity

Phospholipase A2 activity was assayed by three procedures. The first one was the indirect

haemolytic method, an adaptation of the procedures of Habermann and Hardt (8), and Gene et

al. (6); the second was the potentiometric titration (16), and the third, the colorimetric assay

with phenol red (11).

The substrate for the indirect haemolysis was prepared with 0.68 g of agarose A dissolved in

66.5 ml of 50 mM phosphate buffer, pH 7.5, in boiling water bath. The solution was cooled to

52°C, mixed with 1.2 ml of 10 mM CaCl2, 16.5 ml of 3.5 mM egg lecithin, and 1.2 ml of

lamb erythrocytes, and poured into a glass plate (15.4 X 17.6 cm) previously heated to 52°C.

After layer consolidation, cylindrical holes (2.5 mm) were punched. Each hole was filled with

10 µ l of enzyme solution and incubated for 20 hours at 37°C. During incubation, the enzyme

diffused into the gel and cleared the erythrocyte by haemolysis, forming a halo. The diameter

of these areas was measured in millimetres (mm).

Indirect haemolysis assay was used to identify the presence of phospholipase A2 in venom

fractionation and determine its thermal and pH stability. Thermal stability was studied from 6

to 92°C; the enzymatic solution was incubated for 30 minutes, cooled in an ice bath for 5

minutes, and the residual activity was assayed.

The assays for pH stability were performed at pH values ranging from 2.5 to 11.3; the

enzymatic solution was placed in a different buffer for 19 hours at room temperature (18°C),

and the residual activity was assayed.

The potentiometric titration method was used to quantify PLA2 activity in the enzyme

isolation process and in the determination of optimal pH and temperature, using an egg yolk

suspension. The same procedure was used for the determination of the optimal concentration

of Ca2+ and the Michaelis Menten constant (KM), but this time, phosphatidylcholine (Merck)


was used instead of egg yolk. One unit of enzymatic activity was defined as the release of 1.0

meq of fatty acid per min. Specific activity corresponds to the number of meq of fatty acid

liberated per min per mg of protein.

Colorimetric assay was used in order to determine substrate specificity (phosphatidylcholine,

PC; phosphatidylethanolamine, PE; phosphatidylinositol, PD; sphingomyelin, SG; and

cardiolipin, CP). The different substrates were prepared, as described by Lobo de Araujo and

Radvanyi (11), to a final concentration of 0.27% (W/V). One unit of enzymatic activity was

defined as absorbance decrease of 0.01 per min.

RESULTS

Eight fractions of B. asper venom with phospholipase A2 activity were isolated in a two-step

purification procedure. First, the venom was separated by molecular weight on Sephadex G-

75 into five fractions (Figure 1); then, the fraction containing phospholipase activity (F3) was

chromatographied on MONO S (HR5/5) resulting in eight fractions with phospholipase

activity (Figure 2). Table 1 presents the recovery of protein and activity of all fractions

obtained from the two-step purification procedure. F3, resulting from the first step, accounted

for 33% of the venom protein, and the isoenzymes FII and FVII accounted for 10.8% and

8.0% of the protein bounded to the column. PAGE of F3 from the first step purification

procedure showed two bands of 16.2 and 17.2 KDa (Figure 3A); the isoenzymes resulting

from the cation-exchange chromatography migrated as a single band in SDS-PAGE, and their

molecular weights were from 16 to 17 KDa (Figure 3B). These two fractions were the most

interesting since they had more PLA2 activity and protein.

The optimal pH for the FI and FII isoenzymes was 9, and for FVII, 9-10 (Figure 4A), and the

optimal temperature was 52ºC for the three fractions (Figure 4B). The optimal concentration

of Ca2+ for the FII isoenzyme was 48 mM, and for the FVII isoenzyme, it was 384 mM. FII

and FVII showed thermal stability at all the temperatures studied for 30 minutes, and at 92°C

for 60 minutes. The FII isoenzyme was stable at pH values ranging from 2.5 to 9, and FVII

isoenzyme, from 2.5 to 8.


The Michaelis Menten constant (KM) for the FII isoenzyme was 3.5x10-3 M, and for the FVII

isoenzyme, it was 1.6x10-3 M (Figure 5). Kinetic data were analysed by a non-linear model

using the SYSTAT program. The assay of substrate specificity showed the following order:

PC>PD>CP>SG>PE for the FII and FVII isoenzymes. Evaluation of substrate specificity by

the potentiometer method showed no reaction with lysolecithin, confirming that the isolated

enzymes were phospholipases A2 (PLA2s). The FII and FVII isoenzymes did not show any

lethal activity in a dose of 96 and 71.4 µg per 16-18 g mouse.

DISCUSSION

The specific activity of PLA2 found in the venom of B. asper from Chiriguaná was 0.05 U/mg

of protein, similar to other venoms of the Bothrops genus (0.02 mM of fatty acid/min/mg) and

to N. naja venom, which is 0.08 mM of fatty acid/min/mg (12).

The procedure described above for isolation of different fractions with PLA2 activity from the

B. asper venom is simple, quick, and efficient. The first step of purification on gel-filtration

chromatography using 5% of acetic acid facilitated the elution of PLA2 into one fraction (F3).

The total activity of this fraction was 6.12 U, which presented a twofold increase in the

activity in crude venom (3.52 U), as showed in PLA2 from Bothrops insularis (3). F3

represents 33% of the crude venom protein, and has a specific activity of 0.28 U/mg protein,

with a purity factor of 5.28. In other venoms from Bothrops snakes, such as B. insularis (3), it

was found that 6% of the venom protein was PLA2, and for Bothrops lanceolatus alone, 2%

was PLA2 (12). The results showed that in B. asper venom, the proportion of PLA2 is high.

The step of cationic-exchange chromatography using 10 mM of acetic acid facilitated the

elution of the eight fractions with PLA2 from B. asper venom, and showed the multiple forms

of PLA2s present in snake venoms (12,15,23). The isoenzymes found in B. asper were not

dimeric forms, as in other venoms (20,30); however, the same bands are observed in

electrophoresis under non-reducing conditions. PLA2s isolated from B. atrox (15) and

Bothrops n. nummifer (30) were found in the monomeric state, suggested to be the common

state of PLA2 present in Bothrops venoms.


All isoenzymes showed one band in electrophoresis with a molecular weight of 17 KDa,

except for FVII and FVIII (16 KDa). For B. atrox (15) and B. n. nummifer (30), the PLA2s

had a molecular weight of 14 KDa, and for B. lanceolatus (12), this weight was 15 KDa.

Cogo et al. (3) found 4 polypeptide chains between 14 and 17 KDa in the PLA2 fraction from

B. insularis venom. Gutiérrez and Lomonte (7) summarized the molecular weights found for

many myotoxic PLA2s from 10.7 to 16 KDa, in which differences can be observed depending

on the procedure used for the determination. In SDS-PAGE, the molecular weights of some

myotoxic PLA2s were found between 15 and 16 KDa, very close to the values determined for

all the PLA2 fractions isolated in this study.

The optimal pH value for the FI, FII and FVII isoenzymes was 9, at 25°C, very close to those

related as generic for the PLA2 enzymes isolated from other snake venoms (7.5 and 8.5) (9).

The activity is completely inhibited at pH 6 and 12. Salach et al. (24) found 6 isoenzymes in

N. naja venom with optimum pH (7.9 to 8.0) at 25°C, and no significant difference was

observed, except for IIC. This difference may be related to the enzyme conformational change

by pH, as shown by Viljoen et al. (28).

The optimal temperature was different for 3 isoenzymes (FI, FII and FVII), and the maximum

temperature was 52°C. The activities of these three isoenzymes decreased between 10°C and

22°C, and were completely eliminated at 72°C. Nair et al. (18) studied the optimal PLA2

temperatures for different total snake venoms, and found that the venom of several Naja

species (Elapidae) showed their maximum activity at 65°C. In the Viperidae family,

Agkistrodon piscivorus, Bitis gabonica, and Echis carinatus have their maximum activity at

65°C, 55°C, and 50°C, respectively. Besides, several species of Crotalus have their maximum

activity at 45°C. For Trimeresurus flavoviridis venom, it was found that the optimal

temperatures for the three isoenzymes were 40°C, 45°C, and ≥75°C (29).

Determination of the optimal concentration of Ca2+ showed an absolute dependence of this

ion for two of the isoenzymes studied (FII and FVII). Using 4 mM of phosphatidylcholine as

substrate at pH 9 and 52°C, the FII isoenzyme presented maximum activity with 48 mM of

Ca2+, while FVII required 348 mM. These differences in the concentration of Ca2+ are

probably related to the enzyme conformational change produced by this divalent ion (5).


FVII isoenzyme showed high activity with phosphatidylcholine from egg yolk, in contrast to

FI, FII and FVIII assayed by the haemolytic method (results not shown). When egg yolk was

used as substrate in the same method, the maximum activity corresponded to the FI and FII

isoenzymes, progressively decreasing in the other fractions (Figure 2). These results suggest

that one of the differences between the 8 fractions is their substrate specificity, being FVII

highly specific to egg yolk phosphatidylcholine. However, the specific substrate for the FII

and FVII isoforms from B. asper was: PC>PD>CP>SG>PE, being this order different in

PLA2s isolated from other venoms. In T. flavoviridis, the order was PC>PE>Pserina≥PD=0

for three of the isolated isoenzymes (29). The FII and FVII activity on sphingomyelin (SG)

was interesting since that substrate is normally resistant to the action of Phospholipase A2

from several fountains (24,27).

The kinetics constant (KM) showed different responses for the FII and FVII isoenzymes. FII

presented a typical behavior (5), but needed a critical micelle concentration of the substrate

(cmc=1 mM) to initiate its catalytic activity. On the other hand, FVII could act on monomeric

substrates, what suggests that there is not a universal kinetic model for phospholipases or

enzymes with the same origin (6).

The Michaelis Menten constant was 3.5x10-3 and 1.6x10-3 mM for the FII and FVII

isoenzymes, respectively. These KM values are different in PLA2s isolated from other snake

venoms, and were determined using phosphatidylcholine from egg yolk as substrate in the

watery media. The KM value for the PLA2 isolated from C. atrox venom was 7x10-3 M, using

phosphatidylcholine as substrate in the media with diethyl ether (9). For three PLA2

isoenzymes from T. flavoviridis venom, KM values were 2.8x10-6 M, 4.3x10-6 M, and 2.2x10-6

M, using phosphatidylcholine as substrate in the watery media (29).

The two isoenzymes studied did not present any lethal activity for 90 and 71 µg doses when

injected intraperitonially into mice between 16 and 18 g (5.3 µg/g and 4 µg/g), and the crude

venom presented lethal activity for 58 µg. An acid phospholipase A2, isolated from B.

lanceolatus venom, was not toxic when 300 µg were injected into mice (12).


Table 1. Summary of the purification of PLA2 enzymes from B. asper venom.

Step Volume

(ml) Protein*

(mg) Activity**

(U)

Specific Activity (U/mg)

Purity Factor

Venom 1.00 66.00 3.52 0.053 1.00

Sephadex G-75

F1 30.50 9.52 0.00 0.000 0.00

F2 65.50 14.41 0.66 0.046 0.87

F3 90.00 21.85 6.12 0.280 5.28

F4 47.00 3.67 0.00 0.000 0.00

F5 71.00 9.19 0.00 0.000 0.00

TOTAL 304.00 58.64 6.78

Mono S

Fraction F3 *** 85.00 8.50 2.64 0.311 5.87

FI 1.00 0.07 0.12 1.714 32.34

FII 6.20 0.92 1.04 1.130 21.32

FIII 1.54 0.10 0.10 1.000 18.87

FIV 3.90 0.32 0.28 0.875 16.51

FV 3.50 0.16 0.18 1.125 21.23

FVI 3.40 0.11 0.14 1.273 24.02

FVII 4.00 0.68 0.40 0.588 11.09

FVIII 11.50 5.06 0.14 0.028 0.53

TOTAL 35.04 7.42 2.40 * Concentration of protein was estimated by the Lowry et al. method (13).

** The activity of PLA2 was assayed by the titration method (16), using egg yolk in sodium deoxycholate. A unit

of PLA2 enzymatic activity was defined as the quantity of enzyme that releases a meq of fatty acid per

minute.

*** 85 ml of F3 after passing through the Sephadex G-25 column for the buffer change.


Elution volume (ml)

0 100 200 300 400 500 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Abso

rban

ce (2

80nm

)

0

5

10

15

20

25

PLA

2 activity (H

aemolytic diam

eter (mm

)

F1 F2 F3 F4 F5

Figure 1. Sephadex G-75 Column Chromatography of B. asper venom.

100 mg dry weight (66 mg of protein) of B. asper venom was applied to a Sephadex G-75

column (2.6 x 71.5 cm) equilibrated with 5% of acetic acid. The elution was carried out to 60

ml/h with the same solvent and collection fractions of 10 ml. The absorbance was monitored

at 280 nm ( ), and PLA2 activity was assayed by indirect haemolysis ( ) using egg yolk in

saline solution (0.85%).


180 190 200 210 220 230 240 0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

5

10

15

20

25

30

Elution volume (ml)

Abs

orba

nce

( 280

nm

)

PLA

2 activity (H

aemolytic diam

eter (mm

)

FV FVI FVII FVIIIFII FIII FIV FI

Conductivity (m

S)

0

10

20

30

40

50

60

70

Figure 2. Cation-exchange chromatography on mono S of the fraction 3 from Sephadex G-75.

8.5 mg of protein of the fraction 3 (F3) from Sephadex G-75 were applied to the Mono S

column (HR 5/5) equilibrated with 10 mM of acetic acid, pH 5, at 60 ml/h and collection

fractions of 1 ml. The absorbance was monitored at 280 nm ( ); the elution was carried out

in a linear gradient of NaCl concentration from 0 to 1 M, and measured by a conductivity

monitor ( ), PLA2 activity was assayed by indirect haemolysis ( ), using egg yolk in

saline solution (0.85%).


Figure 3. Electrophoresis of the fractions obtained in the isolation of PLA2 from Bothrops

asper snake venom.

Polyacrylamide gel of the PLA2 isolated fractions, in presence of SDS (4%), β-mercaptoethanol (2%),

and nitrate of silver stain. The proteins used as standards were: Phosphorylase B (94,000), Albumin

(67,000), Ovalbumin (43,000), Carbonic Anhydrase (30,000), Trypsin Inhibitor (20,100), α-

lactalbumin (14,400).

A) B. asper venom from Chiriguaná, F1 to F5 separated in chromatography Sephadex G-75. FI, FII

and FVII obtained in the cation-exchange chromatography Mono S.

B) Fraction F3 from the chromatography on Sephadex G-75, FIII to FVIII from cation-exchange

chromatography Mono S. and B. asper venom from Chiriguaná.


Figure 4. Effect of pH and temperature in the reaction velocity of the PLA2 isolated from B.

asper venom.

PLA2 activity was assayed by the Martin-Mountot and Rochat method (16), using an egg yolk suspension as substrate. A) Effect of pH in the reaction velocity. The reaction was carried out at 25ºC and 10 mM of CaCl2. pH was maintained constant by the addition of 5 mM of NaOH. B) Effect of temperature in the reaction velocity. The reaction was carried out at pH 9 (optimum pH) and 10 mM of CaCl2. pH was maintained constant by the addition of 5 mM of NaOH.

J. Ramírez-Avila et al. PURIFICATION AND PARTIAL CHARACTERIZATION OF PHOSPHOLIPASES A2 FROM Bothrops asper (BARBA AMARILLA) SNAKE VENOM FROM CHIRIGUANÁ (CESAR, COLOMBIA). J. Venom. Anim. Toxins incl. Trop. Dis., 2004, 10, 3, p.255

Figure 5. Effect of the substrate concentration in the reaction velocity for the two PLA2s

isolated from B. asper venom.

PLA2 activity was assayed by the Martin-Mountot and Rochat method (16), using

phosphatidylcholine isolated from egg yolk, as substrate. The reaction was carried out in

optimum conditions of pH and temperature (pH 9 and 52ºC), and 100 mM of calcium

chloride. pH was maintained constant by the addition of 5 mM of NaOH. FII isoenzyme with

continuous line ( ) and FVII isoenzyme with discontinues line ( ).

ACKNOWLEDGEMENTS

The authors would like to thank the personnel of Grupo de Sueros: José Arquímedes

González, Luz Losada, and Dioselina Cañón for their contribution; María Cristina Forero,

María Carlina Castillo, and Francisco Ruiz for their support and suggestions. Vladimir

Corredor and Carlos Arturo Hernandez by the revision and comments on the manuscript. This

work was supported by Instituto Nacional de Salud (INS) and Instituto Colombiano para el

Desarrollo de la Ciencia y la Tecnología “Francisco José de Caldas” (COLCIENCIAS),

research grant CT. 217/95.


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